PROJECT SUMMARY Ewing sarcoma is a rare tumor that occurs in children and young adults. While Ewing sarcoma is relatively rare, the biology of this disease is also relevant to non-Ewing sarcomas, and to other cancer subtypes, due to the presence of the EWS/FLI translocation, the driving event in this disease. Similar translocations involving ETS transcription factors occur in other malignancies, including prostate cancer. Thus, understanding the biology of Ewing sarcoma may provide insight into many cancer types. We will investigate the role of secondary genetic events in the pathogenesis of Ewing sarcoma. These secondary events have been identified through next-generation sequencing of primary tumors and cell lines. Specifically, we will evaluate the role of STAG2 in mediating epigenetic and transcriptional changes induced by the driving oncogene in Ewing sarcoma, EWS/FLI. In Aim 1, we will use genomic approaches (RNAseq, ChIPseq, WGS, etc.) to assess the mechanistic role of STAG2 in the regulation of chromatin architecture, transcription, and aneuploidy in Ewing cells. In Aim 2, we will study the role of STAG2 in progression, maintenance and metastasis in Ewing sarcoma, using functional studies in Ewing sarcoma cell lines and patient-derived xenografts. In Aim 3, we will determine whether there is a synthetic lethal dependency on STAG1 when STAG2 is absent in Ewing sarcoma cells. Our central hypothesis is that STAG2 loss plays a key role in the epigenetic reprogramming that drives the oncogenic function of EWS/FLI.